Project 5, T. Ebner: A striking feature of the cerebellar cortex is its two prominent architectures. One is the parasagittal organization of the olivo-cerebellar and cortico-nuclear project, the other is the transversely oriented parallel fibers that run nearly orthogonal to the parasagittal zones. Understanding the function of these two architecture has been hampered by lack of techniques that permit spatial mapping of the activity in the cerebellar cortex. A novel approach using the pH sensitive dye, neutral red, has been developed to spatially map neuronal activation and will be used to image Crus I and II in the rat cerebellar cortex. Experiments are proposed to test the hypothesis that the parasagittal banding pattern represents a map of body surface and stimulus frequency and that activation of the climbing fiber projection is a primary determinant of this parasagittal banding. Experiments are also proposed to test the hypothesis that parallel fibers are activated by peripheral input and contribute to the spatial patterns of activation. Several hypothesis concerning the functional interactions between these two systems will be examined including whether climbing fiber afferents alter the short and/or long term excitability of the responses to surface stimulation and how parallel fibers alter the subsequent responses to peripheral inputs. A limitation of present in vivo optical imaging methodologies is the lack of cell specificity to address this problem lines of transgenic mice have been generated with the gene for cytoplasmic yellow cameleon-2 coupled to the Purkinje cell specific promoter, PCP-2. Yellow cameleon-2 is a Ca++ sensitive fusion protein consisting of two mutant forms of green fluorescence protein linked to Xenopus calmodulin and a 26 residue calmodulin-binding peptide of myosin light chain kinase. Using these transgenic animals, the calcium fluxes occurring in Purkinje cells will be imaged, in vivo, determining the spatial and temporal patterns of Ca++ fluxes evoked by surface, inferior olive, and peripheral stimulation. Overall, these studies will provide a better understanding of how information is processed spatially in the cerebellar cortex.